Molecular modeling and molecular dynamics simulation studies of the GSK3β/ATP/substrate complex: understanding the unique P+4 primed phosphorylation specificity for GSK3β substrates.

Substrate specificity of protein kinases is of fundamental importance for the integrity and fidelity of signaling pathways. Glycogen synthase kinase 3β (GSK3β) has a unique substrate specificity that prefers phosphorylation of its substrates at the P+4 serine before it can further phosphorylate the substrate at the P0 serine in the canonical motif SXXXS(p), where S(p) is the primed phosphorylation site. The detailed phosphorylation mechanism, however, is not clearly understood. In this study, a three-dimensional (3D) model of the ternary complex of GSK3β, ATP, and the phosphorylated glycogen synthase (pGS), termed GSK3β/ATP/pGS, is constructed using a hierarchical approach and by integrating molecular modeling and molecular dynamics (MD) simulations. Based on the 3D model, the substrate primed phosphorylation mechanism is investigated via two 12 ns comparative MD simulations of the GSK3β/ATP/pGS and GSK3β/ATP/GS systems, which differ in the phosphate group bound to the P+4 serine of GS. In agreement with structural analysis, computed binding free energies reveal that the binding of pGS to GSK3β is favored in the prephosphorylated state compared with the GS native state. More importantly, comparison with the system simulated without primed phosphorylation in the GSK3β/ATP/GS complex shows that for an optimal phosphorylation reaction to occur, the pGS priming phosphate in the GSK3β/ATP/pGS system optimizes the proper orientation of the GSK3β N- and C-terminal domains and clamps the P0 serine of pGS in the appropriate configuration for interaction with the ATP γ-phosphate within the catalytic groove.